The hyperglycemia associated with diabetes exacerbates insulin resistance via a direct toxic effect of glucose on skeletal muscle. This in turn contributes to the worsening of glycemic control and the development of morbidity. The mechanism by which glucose exerts a toxic effect on skeletal muscle is unknown, but the end result is decreased sensitivity of the muscle Glut4 glucose transporter to insulin. A line of transgenic mice has been developed in which the constitutive Glut1 glucose transporter is overexpressed specifically in skeletal muscle. Basal glucose transport is dramatically elevated in the muscle of these mice via the constitutive overexpression of Glut1 in the sarcolemma. The increased basal flux of glucose into muscle mimics the effect of hyperglycemia and results in the desensitization of Glut4. These mice provide a unique model system for studying the direct effects of muscle glucose toxicity in vivo in the absence of secondary effects due to hyperglycemia and altered circulating insulin levels. Recent evidence suggests that increased flux of glucose through the glucosamine metabolic pathway may be responsible for glucose- induced insulin resistance in skeletal muscle. The long-term goal of this project is to exploit this transgenic mouse model to help elucidate the mechanism of glucose-induced insulin resistance in skeletal muscle. To accomplish this goal, we propose the following specific aims: l. To determine whether insulin and contraction-induced translocation of Glut4 to the plasma membrane are defective in the muscle of Glut1 transgenic mice using quantitative immunogold electron microscopy. 2. To determine how elevation of intracellular free glucose in vivo affects the pattern of skeletal muscle gene expression and to identify novel genes that may contribute to glucose toxicity. This will be accomplished by the use of reverse transcription differential mRNA display analysis of the Glut1 transgenic skeletal muscle. 3. To determine whether altered glucosamine metabolism is involved in the defect in Glut4 activation in the Glut1 transgenic mice. This will be explored by investigating the direct effect of glucosamine on Glut4 translocation in muscle, measuring levels of glucosamine metabolites in muscle of Glut1 transgenic mice, and examining the effect of glucosamine on the expression of glucose-regulated candidate genes identified in specific aim #2. Transgenic mice overexpressing glutamine:fructose-6- phosphate amidotransferase in muscle will be generated and characterized in order to directly test the hypothesis that increased glucosamine metabolism is responsible for glucose-induced insulin resistance in skeletal muscle.